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J. Biol. Chem., Vol. 276, Issue 28, 25871-25875, July 13, 2001

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Heat Shock RNA Polymerase (E³²) Is Involved in the Transcription of mlc and Crucial for Induction of the Mlc Regulon by Glucose in Escherichia coli^*

Dongwoo Shin, Sangyong Lim^§, Yeong-Jae Seok^¶, and Sangryeol Ryu

From the Research Center for New Bio-Materials in Agriculture, Department of Food Science and Technology and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea and the ^¶ School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Mlc is a global regulator of carbohydrate metabolism. Recent studies have revealed that Mlc is depressed by protein-protein interaction with enzyme IICB^Glc, a glucose-specific permease, which is encoded by ptsG. The mlc gene has been previously known to be transcribed by two promoters, P1(+1) and P2(+13), and have a binding site of its own gene product at +16. However, the mechanism of transcriptional regulation of the gene has not yet been established. In vitro transcription assays of the mlc gene showed that P2 promoter could be recognized by RNA polymerase containing the heat shock sigma factor ³² (E³²) as well as E⁷⁰, while P1 promoter is only recognized by E⁷⁰. The cyclic AMP receptor protein and cyclic AMP complex (CRP·cAMP) increased expression from P2 but showed negative effect on transcription from P1 by E⁷⁰, although it had little effect on transcription from P2 by E³² in vitro. Purified Mlc repressed transcription from both promoters, but with different degrees of inhibition. In vivo transcription assays using wild type and mlc strains indicated that the level of mlc expression was modulated less than 2-fold by glucose in the medium with concerted action of CRP·cAMP and Mlc. A dramatic increase in mlc expression was observed upon heat shock or in cells overexpressing ³², confirming that E³² is involved in the expression of mlc. Induction of ptsG P1 and pts P0 transcription by glucose was also dependent on E³². These results indicate that E³² plays an important role in balancing the relative concentration of Mlc and EIICB^Glc in response to availability of glucose in order to maintain inducibility of the Mlc regulon at high growth temperature.

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